The role of the interactions between an enzyme active-site and its complementary substrate have fascinated biochemists since the initial suggestion of complex formation by Michaelis. The importance of the interactions in the ground state complex have varied from being of absolute importance in a "lock and key" formulation to being "inhibitory" in more recent formulations where interactions in the transition state are the only requirement. An improved understanding of the role of ground state interactions is important both to understanding the general mechanism of enzyme catalysis, and to the de novo design of pharmaceuticals, where enhancing the affinity of a potential pharmaceutical depends on taking advantage of the electronic environment of the active-site. The improvement in spectroscopic instrumentation has allowed the recent determination of the NMR and vibrational spectra of substrates or analogues while bound at the enzyme active site. We have shown that enoyl-CoA hydratase (ECH) is capable of inducing a large polarization of the carbon-carbon double bond that is hydrated during the reaction. The primary goals of this study are to acquire and analyze Raman, 13C and 19F NMR spectra of ECH-bound ligands so that the electronic rearrangement can be quantified, i.e. to use quantum mechanical calculations to model the electron densities at each carbon of the substrate, and to estimate the electronic strain energy in the ligand. Crucially, this provides a new opportunity to directly correlate electronic strain in a Michaelis complex with enhanced chemical reactivity. The introduction of super-notch filters, which efficiently remove Rayleigh scattered photons, and charge coupled device detectors has improved the sensitivity of Raman spectroscopy dramatically. The polarizable enoyl-thiolester substrates utilized generate intense Raman signals, permitting the vibration a frequencies associated with both pi- and sigma-bonds, and hence the electron distribution, in the reactive enoyl-thiolester to be determined. Preliminary spectroscopic studies with para-substituted cinnamoyl-CoA analogs indicate the series incrementally changes the electron density and polarizability of the enoylthiolester. This ability to incrementally control the electron density in the ECH- bound ligand provides the opportunity to quantify the importance of electron density on the carbonyl oxygen to the affinity of the ligand, as well as vibration a properties of the ECH-substrate complex. The methodology developed will be applicable to many enzyme systems, including pharmaceutically important enzymes with thiolester substrates, which include enzymes of unsaturated fatty acid metabolism, steroid and peptide antibiotic biosynthesis and the Mycobacteria tuberculosis enoyl- ACP reductase.